Abstract

Background

Hippocampal demyelination, a common feature of postmortem multiple
sclerosis (MS) brains, reduces neuronal gene expression and is a likely
contributor to the memory impairment that is found in greater than 40% of
individuals with (MS). How demyelination alters neuronal gene expression is
unknown.

Methods

To explore if loss of hippocampal myelin alters expression of
neuronal microRNAs (miRNA), we compared miRNA profiles from myelinated and
demyelinated hippocampi from postmortem MS brains and performed validation
studies.

Findings

A network-based interaction analysis depicts a correlation between
increased neuronal miRNAs and decreased neuronal genes identified in our
previous study. The neuronal miRNA miR-124, was increased in demyelinated MS
hippocampi and targets mRNAs encoding 26 neuronal proteins that were
decreased in demyelinated hippocampus, including the ionotrophic glutamate
receptors, AMPA 2 and AMPA3. Hippocampal demyelination in mice also
increased miR-124, reduced expression of AMPA receptors and decreased memory
performance in water maze tests. Remyelination of the mouse hippocampus
reversed these changes.

Conclusion

We establish here that myelin alters neuronal gene expression and
function by modulating the levels of the neuronal miRNA miR-124. Inhibition
of miR-124 in hippocampal neurons may provide a therapeutic approach to
improve memory performance in MS patients.

Keywords: Multiple sclerosis, myelin, microRNA

Introduction

Multiple Sclerosis (MS) is an inflammatory demyelinating and
neurodegenerative disease of the central nervous system (CNS). MS affects more than
two million people worldwide and over 400,000 individuals in the United States
1,2, where it is the leading cause of non-traumatic neurological
disability in young adults. Greater than 65% of MS patients become cognitively
impaired, with more than 40% having memory dysfunction 3,4. Recently,
there has been increased interest in the role of hippocampal pathology and memory
dysfunction in MS patients 4-7. While levels of neuronal genes are
decreased in demyelinated hippocampus, the underlying mechanisms responsible for
these gene changes remain to be identified. Neuronal genes are both increased and
decreased in demyelinated hippocampi and neuronal loss is modest 5. This implies that demyelination
modulates the transcription and/or translation of neuronal genes.

MicroRNAs (miRNAs) are a class of short, non-protein coding RNAs, capable of
decreasing mRNA translation by binding to 3′ UTR of mRNAs 8. miRNA's are critical regulators of
the development and maturation of neurons and oligodendrocytes 9-12}. Changes in levels of miRNAs and their target genes have been
reported in a variety of neurological diseases including Alzheimer's disease (AD),
Parkinson's disease (PD), Huntington's disease (HD), Tourette's syndrome, and
schizophrenia 13-16. In addition, argonaut, a protein that regulates
the processing of miRNA's is mutated in Fragile-X-syndrome 17. miRNA profiling of white matter
lesions from postmortem MS brains revealed distinct miRNA profiles in active and
inactive demyelinated lesions possibly reflecting increased numbers of infiltrating
immune cells in acute lesions compared to increased reactive gliosis in the chronic
lesions 18. miRNA profiles are
also altered in peripheral blood monocytes from MS patients, but most of the altered
miRNA's differed from those altered in acute or chronic MS brain lesions (reviewed
by 19). Increased levels of miRNAs
in rodent brain can constrain synaptic plasticity and memory function 20-22 and may have similar effects in AD brains 23,24. miRNA changes in myelinated or demyelinated MS hippocampus
have not been reported. Compared to myelinated hippocampus from postmortem MS
brains, we previously reported that demyelinated hippocampi contained a reduction in
both mRNA and protein levels for genes essential for glutamate neurotransmission,
glutamate homeostasis, axonal transport and memory 5. These molecular changes were accompanied by a
significant loss of synaptic density in the demyelinated hippocampus. To investigate
possible mechanisms by which myelin regulates neuronal gene expression, we performed
a comprehensive comparison of miRNAs in myelinated and demyelinated hippocampi from
postmortem MS brains. Our results show that hippocampal demyelination leads to an up
regulation of several neuronal miRNAs including miR-124. Using an animal model of
hippocampal demyelination/remyelination, we show increased miR-124 and reduced mRNA
and protein levels of AMPA receptors in demyelinated hippocampus. In addition, mice
with demyelinated hippocampi had reduced memory performance in water maze tests.
Remyelination returned memory performance and miRNA and AMPA receptor levels to
those observed in control hippocampus. These data highlights how myelin can
influence neuronal gene expression by regulating levels of neuronal miRNA's.

Material and Methods

All postmortem brains were collected as part of the tissue procurement
program approved by the Cleveland Clinic Institutional Review Board. All patient
demographics, tissue processing, RNA isolation, RT-PCR and western blot analysis
have been previously described 5.
MicroRNA arrays were performed by LC Sciences, Houston, TX and the bioinformatic
analysis was performed using iCTNet 25, a plug-in for cytoscape software (www.cytoscape.org). In-situ
hybridization was performed using a modified in situ protocol and LNA-modified
oligonucleotide probes (miRCURY, Exiqon, Denmark). All animal experiments were
performed in strict accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals and were approved by the Institutional Animal
Care and Use Committee of the Cleveland Clinic Foundation using six week old C57BL/6
male mice purchased from Jackson laboratories (Bar Harbor, ME). Full experimental
details are available in the S1 Supplemental Materials and Methods.

Results

Demyelination affects neuronal miRNAs in MS hippocampus

We earlier reported changes in mRNA and protein levels of neuronal genes
following demyelination in MS hippocampus 5. To determine underlying regulatory mechanisms that
could control neuronal gene expression, we compared levels of mature miRNAs in
myelinated and demyelinated hippocampi from postmortem MS brains. Compared to
myelinated hippocampi, 4 miRNAs were significantly decreased and 7 miRNAs were
significantly increased in demyelinated hippocampus (Fig. 1A). Using a bioinformatic approach we
integrated these 11 miRNA's with neuronal specific mRNA transcripts that were
significantly decreased in previous gene profiling comparisons of myelinated and
demyelinated MS hippocampi 5.
For inclusion, these miRNA-mRNA interactions had to be reported in at least 2
independent miRNA target databases. This network analysis show interactions
between the altered miRNA's detected in the present study with the altered
neuronal mRNAs identified in our previous study 5 (Supplemental Fig. 1). To strengthen this
interaction, the miRNA-mRNA network (miRNAs in circles, mRNAs in triangles) was
further merged with protein-protein interactions (retrieved from Human Protein
Reference Database, blue lines) as described previously 25. In this bioinformatic
screening, 9 of the 11 altered miRNAs (miR-24, miR-143, miR-124, miR-30d,
miR-379, miR-138, miR-181a, miR-181c and miR-204) in demyelinated MS hippocampus
show significant association with protein-protein interactions (Supplemental Fig.
2). Among these 9 miRNAs, 5 were increased (miR-24, miR-143,
miR-124, miR-30d, miR-379) and found to be enriched in hippocampal neurons using
fluorescent in situ hybridization.

Representative data shows localization of miR-24 (Fig. 1B-myelinated, ​1C1C-demyelinated) and
miR-30d (Fig. 1D-myelinated,
​1E1E-demyelinated) in MS hippocampus. miRNAs and
their predicted targets that were significantly changed in MS demyelinated
hippocampus are shown in Supplemental Table 1. We also verified the cellular localization of
the target mRNAs in the Allen Brain Atlas, an online resource for mRNA cellular
distribution based upon in situ hybridization (http://www.brain-map.org/). The query showed that 33 out of the 37
target mRNAs were expressed by neurons in rodent brain (Supplemental Table 1).
Interestingly, 26 of these mRNAs contained miR124 binding sites (Supplemental Table 1) in
their 3′UTR region, including the glutamate receptor subunits AMPA1,
AMPA2 and AMPA3. Among the 26 increased mRNAs, 24 were expressed by neurons and
14 were predicted to have a miR-181a binding site (Supplemental Table 1).
These data support the possibility that miR124 and miR181a regulates neuronal
gene expression in demyelinated hippocampal neurons.

Among miRNA's that were increased in demyelinated hippocampi from
postmortem MS brains, miR-124 was the most intriguing as it had 1) the largest
number (26) of predicted neuronal mRNA targets that were significantly decreased
in demyelinated hippocampi from MS brains and 2) increased levels of miR124 have
been correlated with decreased synaptic plasticity and reduce memory performance
in rodents 20,22,23. To
confirm the increase in miR-124 levels in demyelinated hippocampi, we used
RT-PCR and detected a 4.5 fold (p=0.02) increase in miR-124 levels in
demyelinated MS hippocampi compared to myelinated hippocampi (Fig. 2A). Using a combined
immuno-insitu protocol we determined that miR-124 is expressed in myelinated
(Fig. 2B) and
demyelinated (Fig. 2C)
hippocampus and enriched in hippocampal neurons (Fig. 2D-E). Interestingly, miR-124 expression was
confined to neurons in MS hippocampus (Fig
2D-E). Recently, increased miR-124 levels have been
associated with microglial quiescence and suppression of experimental autoimmune
encephalomyelitis in mice 26.
We did not detect miR-124 expression in glial cells in tissue sections from
either myelinated or demyelinated MS hippocampus. We next inquired if loss of
myelin in cortical and sub-cortical white matter regions also leads to increased
levels of miR-124. In control brain tissue, levels of miR-124 were significantly
increased in cortical grey matter compared to sub-cortical white matter
(Fig. 2F) supporting
its neuronal enrichment. The expression of miR-124 in white matter in MS brains
was primarily due to its presence in white matter neurons as shown by
fluorescent in situ hybridization (Fig.
2G). Levels of miR-124 as measured by RT PCR were also
significantly increased in cortical lesions, compared to control or myelinated
MS cortex (Fig. 2F). Similar
to demyelinated hippocampal neurons, miR-124 was enriched in neurons in both
myelinated (Fig. 2H) and
demyelinated MS cortex (Fig.
2I). These studies establish that the neuronal enriched miRNA,
miR-124, is significantly increased in demyelinated hippocampi and cortex from
postmortem MS brains. Despite significant activation of microglia and reactive
astrogliosis in postmortem MS tissue sections, our in situ hybridization studies
failed to detect miR-124 in glial cells. Our data support earlier studies, which
detected low levels of miR-124 in acute or chronic MS white matter lesions
18.

The miRNA changes described above were generated from demyelinated
hippocampi obtained from individuals with a chronic MS disease course. To help
delineate primary vs. secondary neuronal miRNA changes in postmortem MS brains,
we analyzed a mouse model of hippocampal demyelination using dietary cuprizone
combined with intraperitoneal injection of rapamycin. Rapamycin reduces
endogenous remyelination and establishes a more consistent baseline of
demyelination. Compared to rapamycin treatment only, (Fig. 3A), 12 weeks (Fig. 3B) of cuprizone/rapamycin treatment
significantly reduced hippocampal myelin by 95%. Upon returning
cuprizone/rapamycin-treated mice to a normal diet for 6 weeks, remyelination was
prominent (Fig. 3C) and myelin
levels returned to 61% of control levels (Fig. 3D). We next investigated if 12 weeks of
cuprizone/rapamycin treatment leads to decreased memory performance. Using the
Morris water maze test, we examined spatial memory in cuprizone/rapamycin mice.
Rapamycin-treated control mice and cuprizone/rapamycin-treated mice showed
similar latencies in finding a visible platform, supporting normal visual and
motor functions in cuprizone/rapamycin-treated mice. Mice with 12 weeks of
cuprizone/rapamycin treatment took significantly longer times to reach a
submerged platform compared to control mice (Fig. 3E). This spatial memory impairment was reversed in
mice with remyelinated hippocampi (Fig.
3E). These data support the possibility that hippocampal
demyelination is responsible for the memory dysfunction observed in these mice.
We next examined whether miR-124 was increased in demyelinated mouse hippocampi.
Levels of miR-124 was increased 2.6 fold (p=0.038) in demyelinated mouse
hippocampus and returned to control levels following remyelination (Fig. 3F). miR-124 expression was
confined to neurons in control (Fig.
3G), demyelinated (Fig. 3H) and remyelinated (Fig. 3I) rodent hippocampus. The results
establish that demyelination increases expression of the neuronal miRNA, miR-124
and remyelination reverses this change.

Dynamics of miR-124 changes in demyelinated and remyelinated rodent
hippocampus

Given the relationship between miRNA and mRNA in MS hippocampus shown in
Supplemental Fig.
2, we explored the possibility that increased levels of miR-124 could
have a direct regulatory role on mRNA's that encode proteins involved in
synaptic plasticity. Major targets of miRNA-124 include the AMPA glutamate
receptors, GRIA1, GRIA2 and GRIA3. Levels of these three AMPA receptors were
decreased in MS demyelinated hippocampus in postmortem MS brains 5 suggested that increased
expression of miR-124 could lead to reduced levels of AMPA receptors. We next
asked whether AMPA receptors were altered in our rodent hippocampal
demyelination/remyelination model. Levels of the AMPA receptor subunits GRIA1
and GRIA2 were significantly decreased in demyelinated rodent hippocampus
(Fig. 3J). Levels of
GRIA3 were also decreased in mouse hippocampus, but did not reach statistical
significance. Importantly, the levels of these receptors increased upon
remyelination and correlated with decreased levels of miR-124. miRNAs decrease
gene expression by binding to 3′untranslated region (UTR) sequences of
target genes. Sequencing of the 3′ UTR of the three AMPA receptors in
control and MS patients identified miR-124 complementary binding sites
(Supplemental
Fig. 3). We tested whether miR-124 could repress AMPA
receptor expression by placing their 3′UTR segments downstream of a
cytomegalovirus (CMV)-driven luciferase reporter and performed reporter assays
in HEK293 cells transfected with a miR-124 mimic. The presence of miR-124
significantly decreased the luciferase activity of reporters containing the
AMPA1, AMPA2 and AMPA3 3′UTR segments that were predicted to bind miR-124
(Fig. 3K). Mutations
of these miR-124 binding sequences abolished the repressive activities of all
three AMPA receptor luciferase reporters (Fig. 3K). These results support direct binding of miR-124
to the 3′UTR of mRNA encoding the three AMPA receptors. Next we asked if
binding of miR-124 to the 3′UTRs of AMPA receptors cause down-regulation
of AMPA receptor mRNA levels. Transfection of primary neurons with a miR-124
mimic led to a significant (5.4 fold, p=0.004) increase in levels of miR-124
(Supplemental
Fig. 4) and a significant decrease in AMPA1, 2 and 3 mRNA
levels when compared to neurons that were not transfected with the miR-124 mimic
(Fig. 3L). Addition of
a miR-124 inhibitor that blocks endogenous miR-124, however abolished this
decrease and led to a significant increase in AMPA receptor mRNA levels.
Introduction of a scrambled miRNA did not alter AMPA receptor mRNA levels
(Fig. 3L).
Collectively our results indicate that miR-124 binds to and reduces neuronal
AMPA receptor mRNA in primary neuronal cultures. The increase of miR-124
following hippocampal demyelination may therefore play a role in affecting
memory by decreasing levels of AMPA receptors.

In addition to increasing the speed of nerve conduction, myelin plays a
significant role in maintaining the integrity and long-term survival of axons
27. The myelin proteins,
myelin-associated glycoprotein (MAG), proteolipid protein (PLP) and
2′3′-cyclic nucleotide 3′- phosphodiesterase (CNP), play a role
in providing this trophic support and this function appears independent of any role
in myelin sheath formation 27,28. Studies have focused on how the
loss of myelin causes axonal or neuronal degeneration, which is considered the major
cause of permanent neurological disability in primary diseases of myelin (for
reviews, see 27,29,30). From a
mechanistic point of view, recent studies support the transfer of lactate from
oligodendrocytes to axons. This lactate may be a substrate for axonal ATP production
and essential for axonal viability 7,31,32. We propose an additional mechanism whereby myelin
regulates neuronal gene expression by regulating the expression of neuronal miRNA's.
Our studies have leveraged miRNA and mRNA data bases in human and rodent hippocampi
with and without myelin. We identify miR124 as a major negative regulator neuronal
gene expression (26 out of 33) in demyelinated MS hippocampus. miR-124 targets the
3′UTR of AMPA receptors and can decrease AMPA receptor reporter mRNA
expression in in vitro assays. In addition, neuronal miRNA's were
decreased in demyelinated hippocampus and target 3”UTR's of neuronal genes
that are increased in demyelinated hippocampus. miR-181a was significantly decreased
in demyelinated hippocampi and this miRNA targets a majority (14 out of 24) of the
hippocampal neuronal genes (Supplemental Table 1) that were reported to be significantly increased
by demyelination 5. While miRNA's
are regulated by demyelination and remyelination, other neuronal genes decreased in
demyelinated hippocampi 5, such as
KIF1A, do not appear to contain 3” UTR sequences targeted by miRNA's
identified in this study. Demyelination is likely to influence neuronal gene
expression by additional mechanisms that regulate gene transcription. In addition to
the primary effect of demyelination, loss of synapses following demyelination could
also negatively impact expression of synaptic and neuronal genes.

When investigating diseased brain tissue where the proportion of individual
cell types can change, it is imperative to establish which cell type is expressing
individual miRNA's that decrease or increase. For example, if a miRNA is enriched in
oligodendrocytes it would be decreased in MS lesions where oligodendrocytes are
destroyed, but it would have no affect on mRNA translation in that lesion. Similar
concerns could be raised regarding the increase in miR-124 in MS lesions as previous
studies have reported miR-124 controlling activation of microglia 26. Microglia are known to be
activated in some MS lesions. Therefore, we developed a combined
immunocytochemistry-insitu hybridization protocol for identifying cell types
expressing individual miRNAs in rodent and human brain sections. Using this
protocol, miR-124 expression was highly enriched in neuronal cells in both rodent
and human brain and increased in neurons in demyelinated hippocampi and cortex
(Figure 2). Our studies provided a list of
mRNA's that 1) were increased or decreased in demyelinated hippocampus and 2)
contained 3′ UTR sequence targeted by miRNA's that were increased or
decreased in demyelinated hippocampus. Based upon analysis of the Allen in situ
hybridization Brain Atlas (http://www.brain-map.org/), mRNA's that are enriched in neurons are
highlighted in Supplemental Table
1. Therefore, we are confident that the altered miRNA's and mRNA's
reported in Supplemental Table
1 are enriched in neurons.

The role of miR-124 in regulating hippocampal function in MS brains is
supported by studies that correlate increased miR-124 and reduced synaptic
plasticity 21. In addition, a
previous study correlated decreased hippocampal levels of miR-124 with enhanced
memory performance 33 in mice.
These data together with decreased miR-124 expression and increased memory/learning
by remyelination in our rodent model raise the possibility that selective inhibition
of miR-124 in hippocampal neurons could enhance cognitive performance in MS
patients. Minimal neuronal loss in demyelinated MS hippocampus 5 identifies the demyelinated
hippocampal neuron as a viable and abundant therapeutic target.

Supplementary Material

Supp Fig S1-S4

Supp Material

Supp Table S1

Acknowledgements

The authors like to thank Dr. Richard M Ransohoff, MD, Dr. Bruce T Lamb, Ph.D and
Richard M Rudick, MD for helpful comments on the manuscript. The authors would like
to thank Cynthia Swanger, Life Banc for the MS tissue collection program, Olga N.
Kokiko-Cochran for assisting with the rodent behavioral study, Giuxiang Xu for the
primary neuronal cultures and Dr. Christopher Nelson for manuscript editing.

Funding: The work was supported by NMSS RG-4280 (RD), NIH NS35058 and
NIH NS38667 (BDT).

Footnotes

Contributions:

RD designed, conducted the experiments and analyzed the data. AC, MVR,
SAD helped with the in situ hybridization and immunohistochemistry experiments.
AMC, KD, DDE, BB and WBM helped with generation of the mouse model, mouse
injections and behavioral testing. SMS and RJF helped with the procurement of
control and MS tissues. ML and SEB performed the bioinformatic analysis. BDT and
RD wrote the manuscript.